Transformed plants expressing antimicrobial proteins

ABSTRACT

Antimicrobial proteins capable of isolation from seeds of Heuchera or Aesculus show a wide range of antifungal activity and some activity against Gram-positive bacteria. DNA encoding the proteins may be isolated and incorporated into vectors. Plants transformed with this DNA may be produced. The proteins find commercial application as antifungal or antibacterial agents; transformed plants will show increased disease-resistance.

This is a division of application Ser. No. 08/656,318, filed Jun. 12,1996, now U.S. Pat. No. 5,750,504, which is a 371 of PCT/GB94/02766,Dec. 19, 1994.

This invention relates to antimicrobial proteins, processes for theirmanufacture and use, and DNA sequences encoding them. In particular itrelates to antimicrobial proteins capable of being isolated from seedsof Heuchera or Aesculus.

In this context, antimicrobial proteins are defined as proteinspossessing at least one of the following activities: antifungal activity(which may include anti-yeast activity), antibacterial activity.Activity includes a range of antagonistic effects such as partialinhibition or death. Such proteins may be oligomeric or may be singlepeptide subunits.

The genus Heuchera is part of the Saxifragaceae plant family. TheSaxifragaceae is a large widespread family comprising mainly perennialherbs and shrubs, containing the currants and gooseberries as well asmany popular garden flowers.

The genus Aesculus is part of the Hippocastanaceae plant family. TheHippocastanaceae is a small family of trees comprising two genera. Thegenus Aesculus is best known for its ornamental trees, notably the horsechestnut (Aesculus hippocastanum) whose brown seeds are the "conkers"much prized by children.

Plants produce a wide array of antifungal compounds to combat potentialinvaders and over the last ten years it has become clear that proteinswith antifungal activity form an important part of these defences.Several classes of such proteins have been described including thionins,beta-1,3-glucanases, ribosome-inactivating proteins, zeamatins,chitin-binding lectins and chitinases. These proteins have gainedconsiderable attention as they could potentially be used as biocontrolagents.

International Patent Application Number PCT/GB92/01570 (published onMar. 18, 1993 under the publication number WO93/05153, the disclosure ofwhich is incorporated herein by reference) describes a protein classcomprising antifungal proteins (AFPS) and antimicrobial proteins (AMPs).The class includes the following proteins: Rs-AFP1 and Rs-AFP2 capableof isolation from Raphanus sativus (Terras FRG et al, 1992, J Biol Chem,267:15301-13309), Bn-AFP1 and Bn-AFP2 capable of isolation from Brassicanapus, Br-AFP1 and Br-AFP2 capable of isolation from Brassica rapa,Sa-AFP1 and Sa-AFP2 capable of isolation from Sinapis alba, At-AFP1capable of isolation from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2capable of isolation from Dahlia merckii, Cb-AMP1 and Cb-AMP2 capable ofisolation from Cnicus benedictus, Lc-AFP capable of isolation fromLathyrus cicera, Ct-AMP1 and Ct-AMP2 capable of isolation from Clitoriaternatea. This protein class will hereinafter be referred to as "theRs-AFP-type proteins". These and other plant-derived antimicrobialproteins are useful as fungicides or antibiotics, particularly foragricultural purposes. The proteins may be applied to or around a plantor may be expressed within a plant.

We have now purified two new potent antimicrobial proteins.

We have purified a new antimicrobial protein from seeds of Heucherasanguinea, hereinafter called Hs-AFP1 (Heuchera sanguinea--antifungalprotein 1). Hs-AFP1 is a 5 kDa polypeptide; such polypeptides may beassociated as dimers. Hs-AFP1 shows a wide range of antifungal activity.

We have also purified a new antimicrobial protein from seeds of Aesculushippocastanumhereinafter called Ah-AMP1 (Aesculushippocastanum--antimicrobial protein 1). Like Hs-AFP1, Ah-AMP1 is a 5kDa polypeptide. Ah-AMP1 shows a wide range of antifungal activity.

According to the present invention, there is provided an antimicrobialprotein having an amino acid sequence as shown in SEQ ID NO 1 or SEQ IDNO 2, or an amino acid sequence which is substantially homologous to SEQID NO 1 or SEQ ID NO 2 (preferably having at least 60% sequenceidentity) provided that such a protein has antimicrobial activity.

An antimicrobial protein according to the invention is capable of beingisolated from seeds of Heuchera or Aesculus, and may also be capable ofisolation from the seeds of both related and unrelated species, or maybe produced or synthesised by any suitable method.

The antimicrobial protein may be extracted and purified from plantmaterial, manufactured from its known amino acid sequence by chemicalsynthesis using a standard peptide synthesiser, or produced within asuitable organism (for example, a micro-organism or plant) by expressionof recombinant DNA. The antimicrobial protein is useful as a fungicideor an antibiotic and may be used for agricultural or pharmaceuticalapplications.

Amino acid sequencing of Hs-AFP1 and of Ah-AMP1 shows that they arehomologous to the Rs-AFP-type proteins (International Patent ApplicationPublication Number WO93/05153). The amino acid sequence of Hs-AFP1 isshown as SEQ ID NO 1; the amino acid sequence of Ah-AMP1 is shown as SEQID NO 2.

FIG. 5 shows the sequence alignment of Hs-AFP1 (SEQ ID NO 1) and Ah-AMP1(SEQ ID NO 2) with the Rs-AFP-type antifungal/antimicrobial proteinsRs-AFP1 (SEQ ID NO 3), Rs-AFP2 (SEQ ID NO 4), Dm-AMP1 (SEQ ID NO 5),Cb-AMP1 (SEQ ID NO 6), Ct-AMP1 (SEQ ID NO 7) and Lc-AFP (SEQ ID NO 8) asdescribed in International Patent Application Publication NumberWO93/05153. Part of the sequences of the proteins Siα2 from Sorghum (SEQID NO 9) and g1-P from Triticum (SEQ ID NO 10), plus the predicted geneproducts of pSAS10 (Vigna) (SEQ ID NO 11), pI230 (Pisum) (SEQ ID NO 12)and p322 (Solanum) (SEQ ID NO 13) are also shown (discussedhereinafter). Dashes have been introduced into the sequences to giveoptimal alignment.

The Rs-AFP-type proteins share a common structural motif which is alsofound within the sequences of Hs-AFP1 and of Ah-AMP1. Sequence alignmentof Hs-AFP1 and Ah-AMP1 with the Rs-AFP-type proteins shows that theyshare a consensus cysteine-glycine motif which is shown in FIG. 5. It isclear from FIG. 5 that the number of amino acid residues between theconserved cysteines and glycines varies slightly in the differentsequences and partial sequences shown: dashes have been introduced intothe sequences to give optimal alignment. With residue numbering relativeto the sequence of Hs-AFP1, all eight cysteine residues have conservedpositions at residue numbers 6, 17, 23, 27, 39, 48, 50 and 54 and thereare two conserved glycines at position numbers 15 and 37. In addition,there is a conserved aromatic residue at position 13, and a conservedglutamate residue at position 31.

The Hs-AFP1 sequence shows 48% sequence identity with Rs-AFP1. TheAh-AMP1 sequence shows 54% sequence identity with Rs-AFP1. Hs-AFP1 shows52% identity to Ah-AMP1 on the amino acid sequence level. Despite thesimilarities between the Heuchera protein (Hs-AFP1), the Aesculusprotein (Ah-AMP1) and the Rs-AFP-type proteins, there are differences inoverall amino acid content and sequence.

The antifungal activity of Hs-AFP1 causes severe branching(hyper-branching) of fungal hyphae from certain species. Thismorphological effect is similar to that produced by Rs-AFP1 or Rs-AFP2.The protein Ah-AMP1, while inhibiting fungal growth, does not causehyper-branching of hyphae. This activity is more similar to that of theproteins Dm-AMP1, Cb-AMP1, Ct-AMP1 and Lc-AFP.

Hs-AFP1, Ah-AMP1 and the Rs-AFP-type proteins are partially homologousto the predicted protein products of the Fusarium-induced genes pI39 andpI230 in pea (Pisum sativum--a member of the Fabaceae family) asdescribed by Chiang and Hadwiger (1991, Mol Plant Microbe Interact,4:324-331). This homology is shared with the predicted protein productof the pSAS10 gene from cowpea (Vigna unguiculata--another Fabaceae) asdescribed by Ishibashi et al (1990, Plant Mol Biol, 15:59-64). Theproteins are also partially homologous with the predicted proteinproduct of gene p322 in potato (Solanum tuberosum--a member of theSolanaceae family) as described by Stiekema et al (1988, Plant Mol Biol,11:255-269). Recently a protein whose amino-terminal amino acid sequenceis almost identical to the mature protein encoded by p322 has beenpurified from potato tubers and shown to possess antimicrobial activity(Moreno et al, 1994, Eur J Biochem, 233:135-139). Nothing is known aboutthe biological properties of the proteins encoded by genes pI39, pI230,pSAS10 or p322 as only the cDNA has-been studied. However, the pI39 andpI230 genes are switched on after challenge to the plant by a disease orother stress. It has been proposed that the pSAS10 gene encodes aprotein involved in germination.

The Hs-AFP1, Ah-AMP1 and Rs-AFP-type protein sequences show a lowerdegree of partial homology with the sequences of a group of smallα-amylase inhibitors found in the following members of the Gramineae:sorghum (Bloch and Richardson, 1991, FEBS Lett, 279:101-104), wheat(Colitta et al, 1990, FEBS Lett, 270:191-194) and barley (Mendez et al,1990 Eur J Biochem, 194:533-539). Such proteins, including Siα2 fromsorghum and g-1-purothionin (g-1P) from wheat, are known to inhibitinsect α-amylase and may be toxic to insect larvae although this hasnever been shown. It is not known if these α-amylase inhibitors show anyantifungal or other antimicrobial activity: no other data on theirbiological activity has been reported.

Knowledge of its primary structure enables manufacture of theantimicrobial protein, or parts thereof, by chemical synthesis using astandard peptide synthesiser. It also enables production of DNAconstructs encoding the antimicrobial protein.

The invention further provides a DNA sequence encoding an antimicrobialprotein according to the invention. The DNA sequence may be a cDNAsequence or a genomic sequence, and may be derived from a cDNA clone, agenomic DNA clone or DNA manufactured using a standard nucleic acidsynthesiser.

The DNA sequence may be predicted from the known amino acid sequence andDNA encoding the protein may be manufactured using a standard nucleicacid synthesiser. Alternatively, the DNA sequence may be isolated fromplant-derived DNA libraries. Suitable oligonucleotide probes may bederived from the known amino acid sequence and used to screen a cDNAlibrary for cDNA clones encoding some or all of the protein. These sameoligonucleotide probes or cDNA clones may be used to isolate the actualantimicrobial protein gene(s) by screening genomic DNA libraries. Suchgenomic clones may include control sequences operating in the plantgenome. Thus it is also possible to isolate promoter sequences which maybe used to drive expression of the antimicrobial (or other) proteins.These promoters may be particularly responsive to environmentalconditions (such as the presence of a fungal pathogen), and may be usedto drive expression of any target gene.

The DNA sequence encoding the antimicrobial protein may be incorporatedinto a DNA construct or vector in combination with suitable regulatorysequences (promoter, terminator, etc). The DNA sequence may be placedunder the control of a constitutive or an inducible promoter (stimulatedby, for example, environmental conditions, presence of a pathogen,presence of a chemical). Such a DNA construct may be cloned ortransformed into a biological system which allows expression of theencoded protein or an active part of the protein. Suitable biologicalsystems include micro-organisms (for example, bacteria such asEscherichia coli, Pseudomonas and endophytes such as Clavibacter xylisubsp. cynodontis (Cxc); yeast; viruses; bacteriophages; etc), culturedcells (such as insect cells, mammalian cells) and plants. In some cases,the expressed protein may subsequently be extracted and isolated foruse.

An antimicrobial protein according to the invention (such as Hs-AFP1 orAh-AMP1) is useful as a fungicide or an antibiotic. The inventionfurther provides a process of combating fungi or bacteria whereby theyare exposed to an antimicrobial protein according to the invention.

For pharmaceutical applications, the antimicrobial protein may be usedas a fungicide or anti-bacterial to treat mammalian infections (forexample, to combat yeasts such as Candida).

An antimicrobial protein according to the invention may also be used asa preservative (for example, as a food additive).

For agricultural applications, the antimicrobial-protein may be used toimprove the disease-resistance or disease-tolerance of crops eitherduring the life of the plant or for post-harvest crop protection.Pathogens exposed to the proteins are inhibited. The antimicrobialprotein may eradicate a pathogen already established on the plant or mayprotect the plant from future pathogen attack. The eradicant effect ofthe protein is particularly advantageous.

Exposure of a plant pathogen to an antimicrobial protein may be achievedin various ways, for example:

(a) a composition comprising the isolated protein may be applied toplant parts or the surrounding soil using standard agriculturaltechniques (such as spraying); the protein may have been extracted fromplant tissue or chemically synthesised or extracted from micro-organismsgenetically modified to express the protein;

(b) a composition comprising a micro-organism genetically modified toexpress the antimicrobial protein may be applied to a plant or the soilin which a plant grows;

(c) an endophyte genetically modified to express the antimicrobialprotein may be introduced into the plant tissue (for example, via a seedtreatment process);

An endophyte is defined as a micro-organism having the ability to enterinto non-pathogenic endosymbiotic relationships with a plant host. Amethod of endophyte-enhanced protection of plants has been described ina series of patent applications by Crop Genetics InternationalCorporation (for example, International Application Publication NumberWO90/13224, European Patent Publication Number EP-125468-B1,International Application Publication Number WO91/10363, InternationalApplication Publication Number WO87/03303). The endophyte may begenetically modified to produce agricultural chemicals. InternationalPatent Application Publication Number WO94/16076 (ZENECA Limited)describes the use of endophytes which have been genetically modified toexpress a plant-derived antimicrobial protein!.

(d) DNA encoding an antimicrobial protein may be introduced into theplant genome so that the protein is expressed within the plant body (theDNA may be cDNA, genomic DNA or DNA manufactured using a standardnucleic acid synthesiser).

Plant cells may be transformed with recombinant DNA constructs accordingto a variety of known methods (Agrobacterium Ti plasmids,electroporation, microinjection, microprojectile gun, etc). Thetransformed cells may then in suitable cases be regenerated into wholeplants in which the new nuclear material is stably incorporated into thegenome. Both transformed monocotyledonous and dicotyledonous plants maybe obtained in this way, although the latter are usually more easy toregenerate. Some of the progeny of these primary transformants willinherit the recombinant DNA encoding the antimicrobial protein(s).

The invention further provides a plant having improved resistance to afungal or bacterial pathogen and containing recombinant DNA whichexpresses an antimicrobial protein according to the invention. Such aplant may be used as a parent in standard plant breeding crosses todevelop hybrids and lines having improved fungal or bacterialresistance.

Recombinant DNA is heterologous DNA which has been introduced into theplant or its ancestors by transformation. The recombinant DNA encodes anantimicrobial protein expressed for delivery to a site of pathogenattack (such as the leaves). The DNA may encode an active subunit of anantimicrobial protein.

A pathogen may be any fungus or bacterium growing on, in or near theplant. In this context, improved resistance is defined as enhancedtolerance to a fungal or bacterial pathogen when compared to a wild-typeplant. Resistance may vary from a slight increase in tolerance to theeffects of the pathogen (where the pathogen in partially inhibited) tototal resistance so that the plant is unaffected by the presence ofpathogen (where the pathogen is severely inhibited or killed). Anincreased level of resistance against a particular pathogen orresistance against a wider spectrum of pathogens may both constitute animprovement in resistance. Transgenic plants (or plants derivedtherefrom) showing improved resistance are selected following planttransformation or subsequent crossing.

Where the antimicrobial protein is expressed within a transgenic plantor its progeny, the fungus or bacterium is exposed to the protein at thesite of pathogen attack on the plant. In particular, by use ofappropriate gene regulatory sequences, the protein may be produced invivo when and where it will be most effective. For example, the proteinmay be produced within parts of the plant where it is not normallyexpressed in quantity but where disease resistance is important (such asin the leaves).

Examples of genetically modified plants which may be produced includefield crops, cereals, fruit and vegetables such as: canola, sunflower,tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum,tomatoes, mangoes, peaches, apples, pears, strawberries, bananas,melons, potatoes, carrot, lettuce, cabbage, onion.

BRIEF DESCRIPTION OF THE DRAWING

The invention will now be described by way of example only withreference to the drawings, in which:

FIGS. 1A and 1B show the cation exchange chromatogram for purificationof Hs-AFP1 and the associated graph of fungal growth inhibition;

FIGS. 2A and 2B show the reversed phase chromatogram for purifiedHs-AFP1;

FIGS. 3A and 3B show the cation exchange chromatogram for purificationof Ah-AMP1 and the associated graph of fungal growth inhibition;

FIGS. 4A and 4B show the reversed phase chromatogram for purifiedAh-AMP1;

FIG. 5 shows the amino acid sequence alignment of Hs-AFP1, Ah-AMP1 andother proteins; and with reference to the SEQUENCE LISTING in which:

SEQ ID NO 1 is the amino acid sequence of Hs-AFP1;

SEQ ID NO 2 is the amino acid sequence of Ah-AMP1;

SEQ ID NO 3 is the amino acid sequence of Rs-AFP1;

SEQ ID NO 4 is the amino acid sequence of Rs-AFP2;

SEQ ID NO 5 is the amino acid sequence of DM-AMP1;

SEQ ID NO 6 is the amino acid sequence of Cb-AMP1;

SEQ ID NO 7 is the amino acid sequence of Ct-AMP1;

SEQ ID NO 8 is the amino acid sequence of Lc-AFP;

SEQ ID NO 9 is part of the amino acid sequence of Siα2;

SEQ ID NO 10 is part of the amino acid sequence of g1-P;

SEQ ID NO 11 is part of the predicted amino acid sequence of the pSAS10gene product;

SEQ ID NO 12 is part of the predicted amino acid sequence of the pI230gene product;

SEQ ID NO 13 is part of the predicted amino acid sequence of the p322gene product.

EXAMPLE 1 Antifungal and Antibacterial Activity Assays

Antifungal activity was measured by microspectrophotometry as previouslydescribed (Broekaert, 1990, FEMS Microbiol Lett, 69:55-60). Routinely,tests were performed with 20 μl of a (filter-sterilized) test solutionand 80 μl of a suspension of fungal spores (2×10⁴ spores/ml) in halfstrength potato dextrose broth (1/2 PDB). The synthetic growth medium(SMF) consisted of K₂ HPO₄ (2.5 mM), MgSO₄ (50 μM), CaCl₂ (50 μM), FeSO₄(5 μM), CoCl₂ (0.1 μM), CuSO₄ (0.1 μM), Na₂ MoO₄ (2 μM), H₃ BO₃ (0.5μM), KI (0.1 μM), ZnSO₄ (0.5 μM), MnSO₄ (0.1 μM), glucose (10 g/l),asparagine (1 g/l), methionine (20 mg/l), myo-inositol (2 mg/l), biotin(0.2 mg/l), thiamine-HCl (1 mg/l), and pyridoxine-HCl (0.2 mg/l).Control microcultures contained 20 μl of sterile distilled water and 80μl of the fungal spore suspension.

Unless otherwise stated the test organism was Fusarium culmorum (strainIMI 180420) and incubation was done at 25° C. for 48 hours. Percentgrowth inhibition is defined as 100 times the ratio of the correctedabsorbance of the control microculture minus the corrected absorbance ofthe test microculture over the corrected absorbance at 595 nm of thecontrol microculture. The corrected absorbance values equal theabsorbance at 595 nm of the culture measured after 48 hours minus theabsorbance at 595 nm measured after 30 min.

Antibacterial activity was measured microspectrophotometrically asfollows. A bacterial suspension was prepared by inoculating softnutrient agarose (tryptone, 10 g/l; Seaplaque agarose (FMC), 5 g/l).Aliquots (80 μl) of the bacterial suspension (10 colony forming unitsper ml) were added to filter-sterilized samples (20 μl) in flat-bottom96-well microplates. The absorbance at 595 nm of the culture wasmeasured with the aid of a microplate reader after 30 minutes and 24hours of incubation at 28° C. Percent growth inhibition was calculatedas described above for the antifungal activity assay.

EXAMPLE 2 Purification of Antifungal Proteins From Heuchera sanguineaSeeds

Twenty grammes of H sanguinea seeds (obtained from Okkerse, Mechelen,Belgium) was ground in a coffee mill and the resulting meal wasextracted for 2 hours at 4° C. with 100 ml of an ice-cold extractionbuffer containing 10 mM NaH₂ PO₄, 15 mm Na₂ HPO₄, 100 mM KCl, 2 mM EDTA,2 mM thiourea, and 1 mM PMSF. The homogenate was squeezed throughcheesecloth and clarified by centrifugation (30 min at 7,000×g). Thesupernatant was dialyzed extensively against distilled water usingbenzoylated cellulose tubing (Sigma) with a molecular weight cut off of2,000 Da. After dialysis the solution was adjusted to 50 mM (NH₄)Ac (pH9) by addition of the ten-fold concentrated buffer, and subsequentlypassed over a Q-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) column(12×5 cm) in equilibrium with 50 mM NH₄ Ac (pH 9). The protein fractionpassed through the column was lyophilyzed and finally redisolved in 50ml 20 mM NH₄ Ac (ammonium acetate), pH6.

This fraction was applied on a S-Sepharose High Performance (Pharmacia)column (10×1.6 cm) previously equilibrated with 20 mM NH₄ Ac buffer. Thecolumn was eluted at 1 ml\min with a linear gradient of 120 ml from 20to 500 mM NH₄ Ac (pH 6). The eluate was monitored for protein by onlinemeasurement of the absorbance at 280 nm (results shown in FIG. 1B) andcollected in 7.5 ml fractions.

The fractions were lyophilyzed and redissolved in distilled water. Ofthese fractions 5 μl was tested in the microspectrophotometricantifungal activity assays described in Example 1 using the syntheticgrowth medium supplemented with 1 mM CaCl₂ and 50 mM KCl (results shownas histograms in FIG. 1A). All antifungal activity was contained in themajor peak, which eluted at around 300 mM NH₄ Ac (indicated by anarrowhead in FIG. 1B).

The fraction showing highest antifungal activity was further purified byreversed-phase chromatography. This fraction was loaded on a Pep-S(porous silica C₂ /C₁₈, Pharmacia) column (25×0.93 cm) in equilibriumwith 10% acetonitrile containing 0.1% TFA. The column was eluted at 4ml/min with a linear gradient of 200 ml from 10% acetonitrile/0.1%trifluoroacetic acid (TFA) to 95% acetonitrile/0.1% TFA. The eluate wasmonitored for protein by online measurement of the absorption at 280 nm.Two ml fractions of the eluate were collected, vacuum-dried, and finallydissolved in 0.5 ml distilled water of which 10 μl was used in amicrospectrophotometric antifungal activity assay using the syntheticgrowth medium described in Example 1, supplemented with 1 mM CaCl₂ and50 mM KCl.

FIGS. 2A and 2B show the reversed phase chromotogram of the activefraction purified by cation exchange chromatography. FIG. 2B showsmonitoring of the eluate for protein by measurement of the absorbance at280 mm. FIG. 2A shows the antifungal activity as tested by themicrospectrophotometric assay. The chromatogram shows three peaks ofwhich the first, eluting at 25% acetonitrile contains all antifungalactivity. The active factor isolated from this peak (indicated by anarrowhead on FIG. 2B) is called Hs-AFP1 (Heuchera sanguinea--AntifugalProtein 1).

EXAMPLE 3 Purification of Antifungal Protein From Aesculus hippocastanumSeeds

Seeds of Aesculus hippocastanum (Horse Chestnut) were collected fromhorse chestnut trees. The seeds were pealed and 100 g of pealed seedswas lyophilyzed. Lyophilyzed seeds were ground in a coffee mill and theresulting meal was extracted in 100 ml of ice-cold extraction buffer(see Example 1). The homogenate was squeezed through cheesecloth andclarified by centrifugation (30 min at 7,000×g). The supernatant wasdialyzed extensively against distilled water using benzoylated cellulosetubing (Sigma, St Louis, Mo.). After dialysis the solution was adjustedto 50 mM NH₄ Ac (pH 9) by addition of the ten-fold concentrated bufferand passed over a Q-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden)column (12×5 cm) equilibrated in 50 mM NH₄ Ac (pH 9). The proteinfraction which passed through the column was lyophilyzed and finallyredissolved in 50 ml 20 mM NH₄ AC (pH6). This fraction was applied to aS-Sepharose High Performance (Pharmacia) column (10×1.6 cm) equilibratedin 50 mM NH₄ Ac, pH 6.0. The column was eluted at 1 ml\min with a lineargradient of 20-500 ml NH₄ Ac, pH 6.0 over 210 minutes. The eluate wasmonitored for protein by online measurement of the absorbance at 280 nm(results shown in FIG. 3B) and collected in 7.5 ml fractions. Samplesfrom each fraction were lyophilyzed, redissolved in 7.5 ml distilledwater. Of these samples 5 μl was tested in the microspectrophotometricantifungal activity assay using the synthetic growth medium supplementedwith 1 mM CaCl₂ and 50 mM KCl (results shown in FIG. 3A). Followingchromatography, the antifungal activity coeluted with the major peak,which eluted at around 300 mM NH₄ AC (indicated by an arrowhead in FIG.3B). The fraction showing highest antifungal activity was furtherpurified by reversed-phase chromatography. This fraction was loaded on aPEP-S (porous silica C₂ /C₁₈, Pharmacia) column (25×0.4 cm) equilibratedwith 0.1% TFA (trifluoroacetic acid). The column was developed at 1ml/min with a linear gradient of 0.1% TFA to 50% acetonitrile/0.1% TFAover 50 minutes. The eluate was monitored for protein by onlinemeasurement of the absorption at 280 nm (results shown in FIG. 4B). Oneml fractions were collected, vacuum-dried, and dissolved in 0.5 mldistilled water. 5 μl from each fraction was assayed for antifungalactivity (results shown in FIG. 4A). The material yielded a single peakof activity, eluting at 25% acetonitrile. This represents the purifiedprotein Ah-AMP1 (Aesculus hippocastanum--Antifungal Protein 1).

EXAMPLE 4 Molecular Structure of the Purified Antifungal Proteins,Hs-AFP1 and Ah-AMP1

The molecular structure of the purified antifungal proteins was furtheranalysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE). SDS-PAGE was performed on precast commercial gels (PhastGelHigh Density from Pharmacia) using a PhastSystem (Pharmacia)electrophoresis apparatus. The sample buffer contained 200 mM Tris-HCl(pH 8.3), 1% (w/v) SDS, 1 mM EDTA, 0.005% bromophenol blue and, unlessotherwise stated, 1% (w/v) dithioerythritol (DTE). Two hundred nanogramsof the proteins were separated on the gels. Myoglobin fragments wereused as molecular weight markers (Pharmacia) with the following sizes:17 kDa, 14.5 kDa, 8 kDa, 6 kDa, and 2.5 kDa. Proteins were fixed afterelectrophoresis in 12.5% glutaraldehyde and silver-stained according toHeukeshoven and Dernick (1985, Electrophoresis, 6, 103-112).

After reduction of the cysteine residues by DTE, Hs-AFP1 shows a singleband with an apparent molecular mass of about 5 kDa. Unreduced Hs-AFP1migrates as a single band of about 10 kDa. These results show that thenative Hs-AFP1 may be an oligomeric protein, most probably consisting ofa dimer of the 5 kDa polypeptide. The oligomeric structure appears to bestabilised by disulphide linkages.

EXAMPLE 5 Antifungal and Antibacterial Potency

The antifungal potency of the purified proteins was assessed ondifferent plant pathogenic fungi, using the assay described inExample 1. Growth of fungi, collection and harvest of fungal spores andpreparation of mycelial fragments were done as previously described(Broekaert et al, 1990, FEMS Microbiol Lett, 69:55-60). Serial dilutionsof the antifungal proteins were applied to the fungi, either usinggrowth medium SMF- (the synthetic growth medium of Example 1), mediumSMF+ (medium SMF- supplemented with 1 mM CaCl₂ and 50 mM KCl), growthmedium 1/2 PDB- (half strength potato dextrose broth as in Example 1) orgrowth medium 1/2 PDB+ (medium 1/2 PDB- supplemented with 1 mM CaCl₂ and50 mM Kcl). The percent growth inhibition was measured bymicrospectrophotometry. The concentration required for 50% growthinhibition after 48 h of incubation (IC₅₀ value) was calculated from thedose-reponse curves.

The results for Hs-AFP1 and Ah-AMP1 are summarised in Table 1. Theabbreviations used are: IC₅₀ =concentration requred for 50% growthinhibition determined as described in Example 1; ND=not determined;Sp=spores; PrSp=spores pregerminated in the medium for 24 hours;MF=mycelial fragments preincubated for 48 hours in the medium prior toaddition of the proteins; BHR=broad host range; SF=saprophytic fungus;SMF-=synthetic growth medium as described in Example 1; SMF+=SMF-supplemented with 1 mM CaCl₂ and 50 mM KCl; 1/2 PDB-=half strengthpotato dextrose broth as described in Example 1; 1/2 PDB+=1/2 PDB-supplemented with 1 mM CaCl₂ and 50 mM KCl.

                                      TABLE 1    __________________________________________________________________________    ANTIFUNGAL ACTIVITY OF THE HS-AFP1 AND AH-AMP2 PROTEINS                                          PERIOD OF                  STRAIN                        HOST              INCUBATION IN                                                   IC.sub.50 (μg/ml)    FUNGUS        NUMBER                        SPECIES                               MEDIUM                                    INOCULUM                                          AFP-SOLUTION                                                   Ah-AMP1                                                        Hs-AFP1    __________________________________________________________________________    Curvularia affinis                  FAJ97 Musa   SMF- Sp    40       0.75 ND    Curvularia affinis                  FAJ97 Musa   SMF+ Sp    40       25   ND    Gloeosporium musarum                  FAJ86 Musa   SMF- Sp    40       <0.75                                                        ND    Gloeosporium musarum                  FAJ86 Musa   SMF+ Sp    40       12.5 ND    Acremonium hyalinum                  FAJ98 Musa   SMF- Sp    64       <0.75                                                        ND    Acremonium hyalinum                  FAJ98 Musa   SMF+ Sp    64       12.5 ND    Glomerella cingulata                  FAJ76 Musa   SMF- Sp    64       <0.75                                                        ND    Glomerella cingulata                  FAJ76 Musa   SMF+ Sp    64       12.5 ND    Mycoshpaerella fijiensis                  FUL549                        Musa   1/2PDB+                                    MF    96       0.75 12.5    Botrytis cinerea                  K1147 BHR    1/2PDB-                                    Sp    48       25   6    Botrytis cinerea                  K1147 BHR    1/2PDB+                                    Sp    48       >100 25    Alternaria longipes                  CBS620.83                        Nicotiana                               SMF- Sp    70       25   0.75    Alternaria longipes                  CBS620.83                        Nicotiana                               SMF+ Sp    70       100  6    Alternaria brassicae                        Brassica                               SMF- Sp    66       1.5  1.5    Alternaria brassicae                        Brassica                               SMF+ Sp    66       3    3    Alternaria brassicola                  MUCL20297                        Brassica                               SMF- Sp    48       1.5  ND    Alternaria brassicola                  MUCL20297                        Brassica                               SMF+ Sp    48       12   ND    Leptospaeria maculans                  LM36uea                        Brassica                               1/2PDB-                                    Sp    48       0.5  25    Leptospaeria maculans                  LM36uea                        Brassica                               1/2PDB+                                    Sp    48       6    >100    Leptospaeria maculans                  SES1  Brassica                               1/2PDB+                                    PrSp  120      <0.75                                                        >100    Leptospaeria maculans                  SES2  Brassica                               1/2PDB+                                    PrSp  120      <0.75                                                        >100    Cercospora beticola                  SES   Beta   1/2PDB+                                    PrSp  72       1.5  1.5    Fusarium culmorum                  IMI180420                        Cereals                               SMF- Sp    48       1.5  0.5    Fusarium culmorum                  IMI180420                        Cereals                               SMF+ Sp    48       25   1    Fusarium culmorum                  KO311 Cereals                               1/2PDB-                                    Sp    48       12   1    Fusarium culmorum                  KO311 Cereals                               1/2PDB+                                    Sp    48       >100 3    Septoria tritici                  K1097 Triticum                               1/2PDB-                                    Sp    48       0.5  0.5    Septoria tritici                  K1097 Triticum                               1/2PDB+                                    Sp    48       1.5  3    Ascochyta pisi                  MUCL30164                        Pisum  SMF- Sp    48       0.8  1.5    Ascochyta pisi                  MUCL30164                        Pisum  SMF+ Sp    48       15   >5    Penicillum digitatum                  K0879 Citrus fruit                               1/2PDB-                                    Sp    48       6    1    Penicillum digitatum                  K0879 Citrus fruit                               1/2PDB+                                    Sp    48       25   3    Verticillium albo-atrum                  K0937 Lycoperisicum                               1/2PDB-                                    Sp    48       6    12    Verticillium albo-atrum                  K0937 Lycoperisicum                               1/2PDB+                                    Sp    48       >100 30    Cladosporium sphaerospermum                  K0791 SF     1/2PDB-                                    Sp    48       0.5  1    Cladosporium sphaerospermum                  K0791 SF     1/2PDB+                                    Sp    48       12   3    Trichoderma viride                  K1127 SF     1/2PDB-                                    Sp    48       >100 15    Trichoderma viride                  K1127 SF     1/2PDB+                                    Sp    48       >100 >100    __________________________________________________________________________

The results in Table 1 illustrate the wide range of antifungal activitypossessed by the Ah-AMP1 and Hs-AFP1 proteins. The results in Table 1also show that Hs-AFP1 and Ah-AMP1 have a somewhat different activityspectrum. For example, Ah-AMP1 is highly active in 1/2 PDB+ onLeptospaeria maculans but Hs-AFP1 is not, whereas Hs-AFP1 is highlyactive in 1/2 PDB+ on Fusarium culmorum but Ah-AMP1 is not. The twoproteins may thus be used to complement eachother as a combinedantimicrobial agent, combatting a wider range of diseases moreeffectively.

In media SMF+ and 1/2 PDB+ (containing salt additives to reflect theionic strength of physiological conditions in plant tissues), theactivity of both proteins is reduced compared to their activity in mediaSMF- or 1/2 PDB-. The salt-dependent activity reduction is clearlydependent on the test organism. For example, the reduction of activityin medium SMF- versus SMF+ for Ah-AMP1 is two-fold on Alternariabrassicae and more than ten-fold on Gloeosporium musarum.

Hs-AFP1 and Ah-AMP1 both interfere with fungal growth processes, shownby a reduction in hyphal length. Hs-AFP1 also causes hyper-branching ofhyphae of Fusarium culmorum. A similar effect is produced by the theantifungal proteins Rs-AFP1 and Rs-AFP2 from Raphanus sativus seeds(Terras FRG et al, 1992, J Biol Chem, 267:15301-15309). Ah-AMP1 does notcause hyper-branching of Fusarium culmorum hyphae; its mode of hyphalgrowth inhibition is very similar to that of the antifungal proteinsDM-AMP1, Cb-AMP1, Ct-AMP1 and Lc-AFP from Dahlia, Cnicus, Clitorea andLathyrus respectively (International Patent Application PublicationNumber WO93/05153).

The antibacterial activities of AH-AMP1 and Hs-AFP1 were measured onfour gram positive bacteria (Bacillus subtilis JHCC 553331; Micrococcusluteus ATCC 93411; Staphylococcus aureus ATCC 25923; Streptococcusfeacolis ATCC 29212) and two gram negative bacteria (Eschericia coliHB101 and Proteus vulgaris JHCC 558711). Hs-AFP1 did not inhibit thegrowth of these bacteria tested at rates of 200 μg/ml, whereas Ah-AMP1inhibited growth of B subtilis at 100 μg/ml.

EXAMPLE 6 Inhibition of α-amylase

A crude α-amylase extract was prepared from dissected guts of adultcockroaches (Blatta orientalis) by homogenising in 20 mM Tris/HCl, pH7.5, 10 mM CaCl₂ and removal of cell-debris by centrifugation. Human andporcine α-amylases were purchased from Sigma. Type 1-α-amylase inhibitorfrom wheat (purchased from Sigma) was used as a positive control.Amylase extracts were incubated with peptides for 20 min at 30° C. priorto addition of starch and enzyme activity was detected using the methodof Bernfeld (1955, Methods Enzymol, Colwick and Kaplan eds, vol1:149-158).

The α-amylase inhibition activities of Hs-AFP1 and AH-AMP1 were comparedon α-amylases from the three sources to that of the Sorgum bicolorhomologue SIα3, previously reported to inhibit insect gut α-amylases(Bloch and Richardson, 1991, FEBS Lett, 279:101-104). SIα3 inhibited theactivity of the enzymes from insect gut and human saliva to greater than70% at rates as low as 5 μg/ml. Comparable inhibition was achieved with10 U/ml of a commercial preparation of type 1 α-amylase inhibitor fromwheat. SIα3 was essentially inactive on the enzyme from porcine pancreasas previously reported by Bloch and Richardson (1991).

In contrast, no inhibition of α-amylase activity was observed withHs-AFP1 or Ah-AMP1 tested on the three enzymes even when included atrates as high as 200 μg/ml.

EXAMPLE 7 Amino Acid Sequencing of Hs-AFP1 and Ah-AMP1

Cysteine residues of the antifungal proteins were modified byS-pyridylethylation using the method of Fullmer (1984, Anal Biochem,142, 336-341). Reagents were removed by HPLC on a Pep-S (porous silicaC₂ /C₁₈) (Pharmacia) column (25×0.4 cm). The S-pyridylethylated proteinswere recovered by eluting the column with a linear gradient from 0.1%trifluoroacetic acid (TFA) to acetonitrile containing 0.1% TFA. Theresulting protein fractions were subjected to amino acid sequenceanalysis in a 477A Protein Sequence (Applied Biosystems) with on-linedetection of phenylthiohydantoin amino acid derivatives in a 120AAnalyser (Applied Biosystems).

The amino acid sequence of Hs-AFP1 and Ah-AMP1 were determined byN-terminal automated Edman degradation. The complete amino acid sequencefor Hs-AFP1 is given as SEQ ID NO 1, and the complete amino acidsequence for Ah-AMP1 is given as SEQ ID NO 2.

Hs-AFP1 is 54 amino acids in length, and Ah-AMP1 is 50 amino acids inlength. Both proteins contain eight cysteines and basic amino acidresidues are relatively abundant.

Hs-AFP1 contains a tyrosine residue at position 41, a phenylalanineresidue at position 43 and a proline residue at position 44. Identicalamino acids are found in corresponding positions of Rs-AFP1 and Rs-AFP2(Y at position 38, F at position 40, P at position 41), but are replacedby non-homologous amino acid residues in Ah-AMP1 and the otherRs-AFP-type proteins. A study of the 3-dimensional folding of a relatedprotein from wheat seeds (Bruix et al, 1993, Biochemistry, 32:715-724)indicates that these conserved amino acids are located on a protein loopconnecting two antiparallel β-sheets. This loop may be part of theactive site that is responsible for interaction with putativereceptor(s) on fungal hyphae.

It is noteworthy that Rs-AFP1, Rs-AFP2 and Hs-AFP1 all causehyperbranching of fungi, whereas Ah-AMP1, Dm-AMP1, Dm-AMP2, Cb-AMP1 andCb-AMP2 do not. This indicates that each group of proteins may beinteracting either with a different target in the fungus or with thesame target in a distinct way.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 13    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 54 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Hs-AFPl    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Asp Gly Val Lys Leu Cys Asp Val Pro Ser Gl - #y Thr Trp Ser Gly His    #                15    - Cys Gly Ser Ser Ser Lys Cys Ser Gln Gln Cy - #s Lys Asp Arg Glu His    #            30    - Phe Ala Tyr Gly Gly Ala Cys His Tyr Gln Ph - #e Pro Ser Val Lys Cys    #        45    - Phe Cys Lys Arg Gln Cys        50    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Ah-AMP1    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Leu Cys Asn Glu Arg Pro Ser Gln Thr Trp Se - #r Gly Asn Cys Gly Asn    #                15    - Thr Ala His Cys Asp Lys Gln Cys Gln Asp Tr - #p Glu Lys Ala Ser His    #            30    - Gly Ala Cys His Lys Arg Glu Asn His Trp Ly - #s Cys Phe Cys Tyr Phe    #        45    - Asn Cys        50    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 51 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Rs-AFP1    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Glx Lys Leu Cys Glu Arg Pro Ser Gly Thr Tr - #p Ser Gly Val Cys Gly    #                15    - Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile As - #n Leu Glu Lys Ala Arg    #            30    - His Gly Ser Cys Asn Tyr Val Phe Pro Ala Hi - #s Lys Cys Ile Cys Tyr    #        45    - Phe Pro Cys        50    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 51 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Rs-AFP2    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Glx Lys Leu Cys Gln Arg Pro Ser Gly Thr Tr - #p Ser Gly Val Cys Gly    #                15    - Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Ar - #g Leu Glu Lys Ala Arg    #            30    - His Gly Ser Cys Asn Tyr Val Phe Pro Ala Hi - #s Lys Cys Ile Cys Tyr    #        45    - Phe Pro Cys        50    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Dm-AMPl    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Glu Leu Cys Glu Lys Ala Ser Lys Thr Trp Se - #r Gly Asn Cys Gly Asn    #                15    - Thr Gly His Cys Asp Asn Gln Cys Lys Ser Tr - #p Glu Gly Ala Ala His    #            30    - Gly Ala Cys His Val Arg Asn Gly Lys His Me - #t Cys Phe Cys Tyr Phe    #        45    - Asn Cys        50    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Cb-AMP1    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Glu Leu Cys Glu Lys Ala Ser Lys Thr Trp Se - #r Gly Asn Cys Gly Asn    #                15    - Thr Lys His Cys Asp Asp Gln Cys Lys Ser Tr - #p Glu Gly Ala Ala His    #            30    - Gly Ala Cys His Val Arg Asn Gly Lys His Me - #t Cys Phe Cys Tyr Phe    #        45    - Asn Cys        50    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 49 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Cb-AMP1    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Asn Leu Cys Glu Arg Ala Ser Leu Thr Trp Th - #r Gly Asn Cys Gly Asn    #                15    - Thr Gly His Cys Asp Thr Gln Cys Arg Asn Tr - #p Glu Ser Ala Lys His    #            30    - Gly Ala Cys His Lys Arg Gly Asn Trp Lys Cy - #s Phe Cys Tyr Phe Asp    #       45    - Cys    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: llnear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Lc-AFP    #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Lys Thr Cys Glu Asn Leu Ser Gly Thr Phe Ly - #s Gly Pro Cys Ile Pro    #                15    - Asp Gly Asn Cys Asn Lys His Cys Lys Asn As - #n Glu His Leu Leu Ser    #            30    - Gly Arg Cys Arg Asp Asp Phe Xaa Cys Trp Cy - #s Thr Arg Asn Cys    #        45    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: pSAS10    #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Lys Thr Cys Glu Asn Leu Val Asp Thr Tyr Ar - #g Gly Pro Cys Phe Thr    #                15    - Thr Gly Ser Cys Asp Asp His Cys Lys Asn Ly - #s Glu His Leu Leu Ser    #            30    - Gly Arg Cys Arg Asp Asp Val Arg Cys Trp Cy - #s Thr Arg Asn Cys    #       45    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: pI230    #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Asn Thr Cys Glu Asn Leu Ala Gly Ser Tyr Ly - #s Gly Val Cys Phe Gly    #                15    - Gly Cys Asp Arg His Cys Arg Thr Gln Glu Gl - #y Ala Ile Ser Gly Arg    #            30    - Cys Arg Asp Asp Phe Arg Cys Trp Cys Thr Ly - #s Asn Cys    #        45    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 48 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Sia3    #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Arg Val Cys Met Gly Lys Ser Ala Gly Phe Ly - #s Gly Leu Cys Met Arg    #                15    - Asp Gln Asn Cys Ala Gln Val Cys Leu Gln Gl - #u Gly Trp Gly Gly Gly    #            30    - Asn Cys Asp Gly Val Met Arg Gln Cys Lys Cy - #s Ile Arg Gln Cys Trp    #        45    - (2) INFORMATION FOR SEQ ID NO: 12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    - (iij MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: ylP    #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Lys Ile Cys Arg Arg Arg Ser Ala Gly Phe Ly - #s Gly Pro Cys Met Ser    #                15    - Asn Lys Asn Cys Ala Gln Val Cys Gln Gln Gl - #u Gly Trp Gly Gly Gly    #            30    - Asn Cys Asp Gly Pro Phe Arg Arg Cys Lys Cy - #s Ile Arg Gln Cys    #        45    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: p322    #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Arg His Cys Glu Ser Leu Ser His Arg Phe Ly - #s Gly Pro Cys Thr Arg    #                15    - Asp Ser Asn Cys Ala Ser Val Cys Glu Thr Gl - #u Arg Phe Ser Gly Gly    #            30    - Asn Cys His Gly Phe Arg Arg Arg Cys Phe Cy - #s Thr Lys Pro Cys    #        45    __________________________________________________________________________

We claim:
 1. An isolated DNA encoding a protein having an amino acidsequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO:
 2. 2. The isolated DNA as claimed in claim 1, wherein the proteinhas an amino acid sequence of SEQ ID NO:
 1. 3. The isolated DNA asclaimed in claim 1, wherein the protein has an amino acid sequence ofSEQ ID NO:
 2. 4. A transformed biological system containing DNA asclaimed in claim
 1. 5. A transformed biological system as claimed inclaim 4 which is a micro-organism.
 6. A transformed biological system asclaimed in claim 4 which is a plant.
 7. A transformed plant containingthe isolated DNA as claimed in claim
 1. 8. A process of producingprotein comprising expressing the isolated DNA as claimed in claim 1under control of a constituitive or an inducible promoter.